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https://tkuir.lib.tku.edu.tw/dspace/handle/987654321/32649
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| 题名: | 酵母菌IDS2基因在大腸桿菌中的蛋白質表現和純化以及甲基化活性 |
| 其它题名: | Expression, purification, and methylation activity of yeast IDS2p in escherichia coli |
| 作者: | 林榆清;Lin, Yu-chin |
| 贡献者: | 淡江大學生命科學研究所碩士班
陳銘凱;Chern, Ming-kai |
| 关键词: | IDS2;啤酒酵母菌;甲基轉移酶;IEF;IDS2;ISaccharomyces cerevisiae;methyltransferase;IEF |
| 日期: | 2009 |
| 上传时间: | 2010-01-11 02:30:47 (UTC+8) |
| 摘要: | 蛋白質會由許多種方式來修飾，其中一種為蛋白質的甲基化，而形成蛋白質甲基化的酵素為甲基轉移酶，甲基轉移酶是將S-adenosylmethionine (S-腺甲硫氨酸)所提供的甲基轉移到蛋白質上。蛋白質的甲基化作用是一種轉譯後修飾常發生於polypeptide chain，它會調節生物的生理作用，包括DNA、RNA的代謝、蛋白質的生合成及訊息傳導。
Ime2p是只在啤酒酵母菌（Saccharomyces cerevisiae）進行減數分裂時才會表現的protein kinase，而Ime2p會刺激早期、中期、晚期的減數分裂基因的表現，IDS2則是刺激Ime2p作用的基因,兩者之間在功能上相互影響。
而根據以往文獻的報導，IDS2p具有甲基轉移酶之特徵序列。我們將啤酒酵母菌（Saccharomyces cerevisiae）IDS2基因建構到大腸桿菌的Rosseta 菌株中作蛋白質表現，利用His-tag融合純化出所需要的IDS2p，並及將△IDS2菌株之蛋白質抽取液作受質，加入氚化甲基S-腺苷甲硫氨酸[C3H3]S-adenosy-L-methionine一起進行甲基化反應，接著將受質蛋白質加以分離。將受質蛋白質以直立式凝膠電泳isoelectric focusing (IEF)進行第一維的分離,然後將分離的受質蛋白質取下進行SDS-PAGE的第二維電泳，最後我們將分離出的受質蛋白質取下進行質譜儀的鑑定分析。
Proteins can be modified in several ways by the addition of methyl groups from S-adenosylmethionine. Methylation regulates a variety of biological functions including DNA and RNA metabolism, protein synthesis and signal transduction.
Ime2p is a protein kinase that is expressed only during meiosis in Saccharomyces cerevisiae. Ime2p stimulates early, middle, and late meiotic gene expression. IDS2 (for IME2-dependent signaling) has a functional interaction with Ime2p.
According to recent studies, the sequence IDS2, is homologous to a methyltransferase. In our studies, we constructed the sequence IDS2 of Saccharomyces cerevisiae and overexpressed IDS2p in E. coli Rosetta (DE3) strains, and purified IDS2p with His-tag. The protein extract from △IDS2 strain was methylated with the cosubstrate [C3H3]S-adenosy-L-methionine prior to separation. The labeled proteins were first separated by isoelectric focusing (IEF) on a vertical slab gel, and we cutted off the spot with labeled protein and separated the protein on SDS-PAGE. Finally the labeled protein was extracted from SDS-PAGE and identified by mass spectrometry. |
| 显示于类别: | [生命科學研究所] 學位論文
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